Clinical examination and diagnosis of diseases are performed by detecting genes and proteins related to the diseases which are contained in biological samples by detection methods such as a gene detection method and an immunological detection method. Examples of the detection methods include immunochromatography, latex agglutination, enzyme immunoassay, chemiluminescent immunoassay, and PCR assay, and the like.
However, these detection methods have room for improvement in detectability, quantitative performance, measuring range, and the simplicity and rapidity of operations.
A method of using an electric current generated by photoexcitation of a photochemically active labeling substance and light or an electric current generated by applying a voltage of an electrochemically active labeling substance to detect analytes such as genes and proteins (electrochemical detection method) is proposed for the purpose of improving detection sensitivity, quantitative performance, and simplicity (see, for example, U.S. Patent Publication No. 2009/294305, U.S. Pat. Nos. 5,776,672, 5,972,692, U.S. Patent Publication No. 2010/108539, U.S. Patent Publication No. 2010/112578, and U.S. Patent Publication No. 2003/080284).
U.S. Patent Publication No. 2009/294305 describes a method of detecting an analyte, comprising: irradiating the analyte labeled with a photochemically active sensitizing dye with light; and measuring an electric current caused by photoexcitation of the sensitizing dye contained in the labeled analyte (hereinafter referred to as “photoelectrochemical detection”). In the method described in U.S. Patent Publication No. 2009/294305, the labeled analyte is brought into contact with a working electrode containing a trapping substance capable of binding directly or indirectly to the label analyte on the surface. Thus, the labeled analyte is immobilized on the working electrode through the trapping substance. Subsequently, the working electrode and a counter electrode are brought into contact with an electrolyte medium, and the labeled analyte immobilized on the working electrode is irradiated with light to excite the sensitizing dye. Thereafter, the analyte is specifically detected by measuring a photocurrent which flows between the working electrode and the counter electrode due to electronic transition from the photoexcited sensitizing dye to the working electrode.
U.S. Pat. Nos. 5,776,672 and 5,972,692 disclose gene detection methods using a single-stranded nucleic acid probe having a base sequence complementary to the base sequence of a target gene which is immobilized on an electrode and a double-stranded nucleic acid recognizing substance which specifically binds to double strand nucleic acid and contains a labeling substance which is electrochemically active. In the methods described in U.S. Pat. Nos. 5,776,672 and 5,972,692, a sample containing nucleic acid that is denatured into a single strand, a probe, the double-stranded nucleic acid recognizing substance are contacted with one another. Then, a target gene is detected by measuring an oxidation reduction current or electrochemical luminescence based on the labeling substance contained in the double-stranded recognizing substance bound to a double strand nucleic acid which is formed by hybridization between a nucleic acid corresponding to the target gene and a probe.
Methods of electrochemically detecting an analyte using a test chip which includes a working electrode having a conductive layer and an electron accepting layer, a probe immobilized on the electron accepting layer, a counter electrode, and a reduction electrode are described in U.S. Patent Publication No. 2010/108539 and U.S. Patent Publication No. 2010/112578. In the methods described in Publication No. 2010/108539 and U.S. Patent Publication No. 2010/112578, an electrolytic reduction reaction is facilitated by applying a potential to the reduction electrode of the test chip. Accordingly, the current flowing between the working electrode and the counter electrode is increased and the detectability of analyte is improved.
A method including allowing an analyte labeled with a labeling substance to be trapped by a probe immobilized on a metal layer formed on a semiconductor layer of the working electrode and electrochemically detecting it is described in U.S. Patent Publication No. 2003/080284. In the method described in U.S. Patent Publication No. 2003/080284, the analyte labeled with the labeling substance is first trapped by the probe immobilized on the metal layer formed on the semiconductor layer of the working electrode. Thereafter, the metal layer is dissolved. Then, the photocurrent flowing between the working electrode and the counter electrode is detected.
In these detection methods, the analyte is detected through a labeling substance which is electrochemically or photochemically active. Therefore, the working electrode in which a trapping substance for trapping an analyte is immobilized on the surface so that the labeling substance is present near the working electrode according to the amount of the analyte has been used.
However, the methods described in U.S. Patent Publication No. 2009/294305, U.S. Pat. Nos. 5,776,672, 5,972,692, U.S. Patent Publication No. 2010/108539, and U.S. Patent Publication No. 2010/112578 have the following defects.
The first defect is that noise can occur. This is because the trapping and detection regions of the analyte are located in the same region and impurities present in a sample are nonspecifically adsorbed on the working electrode.
The second defect is that when a large-sized analyte is to be detected, the detection capacity can be reduced. This is because it is difficult to make the labeling substance present near the working electrode due to steric interference and the distance between the working electrode and the labeling substance becomes larger.
The third defect is that the reuse of the working electrode with the trapping substance immobilized may be difficult. This is because, in order to reuse the working electrode with the trapping substance immobilized, it is necessary to remove substances other than the trapping substance on the working electrode by a cleaning process. In this case, the trapping substance may also be removed from the working electrode in the cleaning process. Further, the trapping substance on the working electrode may be denatured by a cleaning agent to be used in the cleaning process. Thus, the time of reusing the working electrode with the trapping substance immobilized may affect the measurement results. Therefore, a detection unit which includes the working electrode with the trapping substance immobilized is usually thrown away for each measurement process. Consequently, a detection system using the working electrode with the trapping substance immobilized causes higher costs per measurement.
The second defect is improved by the method described in U.S. Patent Publication No. 2003/080284. However, the first and third defects are remained. As for the method described in U.S. Patent Publication No. 2003/080284, in the process of allowing the analyte to be trapped by the probe, the metal layer may be peeled off depending on conditions.
There is suggested a process of forming an adhesion layer made of titanium, palladium or chromium as an intermediate layer between the working electrode and the metal layer in order to improve the adhesion between the working electrode and the metal layer and prevent the metal layer from being peeled off. However, in this case, the photocurrent generated from the adhesion layer can be noise.